Tolerogenic fusion proteins of immunoglobulins and methods for inducing and maintaining tolerance

ABSTRACT

The invention provides methods and compositions for inducing and maintaining tolerance to epitopes or antigens containing the epitopes. The compositions include expression cassettes and vectors including DNA sequences coding for a fusion immunoglobulin operably linked to transcriptional and translational control regions functional in a hemopoietic or lymphoid cell. The fusion immunoglobulin includes at least one heterologous tolerogenic epitope at the N-terminus variable region of the immunoglobulin. Cells stably transformed with the expression vector are formed and used to produce fusion immunoglobulin. The invention also provides methods for screening for novel tolerogenic epitopes and for inducing and maintaining tolerance. The methods of the invention are useful in the diagnosis and treatment of autoimmune or allergic immune responses.

BACKGROUND OF THE INVENTION

[0001] Self-nonself discrimination is one of the cornerstones ofimmunology. Normally, individuals develop tolerance to self constituentsduring the early development of the immune system. However, themaintenance of this unresponsive state requires the persistence ofantigen, a fact which implies that tolerance induction is a lifelongprocess. Smith, Advances in Immunology, 1:67 (1961). Indeed, thebreakdown of tolerance in older individuals explains the increasedincidence of autoimmunity in aging populations.

[0002] Isologous or heterologous gamma globulins have been used astolerogenic carrier molecules (primarily IgG's). Scott, Immunol. Rev.,43:241 (1979). Although different sources of IgG's may vary in theirpersistence and/or mechanism of tolerance induction, by far, IgGcarriers have been the most efficacious at tolerance induction in adultsto haptens, nucleosides and peptides. Borel, Immunological Reviews,50:71 (1980); and Scott, Cell Immunol., 22:311 (1976). These carriersowe their superior tolerogenicity to their persistence in vivo and theability of epitopes chemically attached to IgG's to crosslink mIgM withB-cell Fc receptors. However, chemical crosslinking of epitopes to IgGcarriers is limited by the availability of free amino groups and theuncontrolled targeting of the added determinant to different portions ofthe IgG.

[0003] Recombinant DNA technology can be used to genetically engineermolecules having heterologous epitopes. For example, heterologousoligopeptide epitopes of biological interest have been expressed inbacterial flagellin (Jennings et al., Protein Eng., 2:365 (1989));hepatitis B surface antigen (Rutgers et al., Biotechnology, 6:1065(1988)); and in the complementarity determining regions ofimmunoglobulins (Zanetti et al., Nature, 355:476 (1992). Some attemptshave been made to test the ability of recombinant proteins to serve asantigens to immunize animals and generate immune responses to theheterologous oligopeptide. However, induction and maintenance oftolerance to oligopeptides presented to the immune system has not beendemonstrated. The ability to maintain tolerance to an antigen or epitoperequires persistence of the epitope in vivo.

[0004] Therefore, there is a need to develop a method of inducing stableand long lasting tolerance to an epitope. There is a need to develop avector that can provide for persistence of the epitope in vivo so thattolerance is maintained. There is a need to develop a recombinant vectorwhich codes for a recombinant polypeptide that has a heterologousepitope and that can be used to induce and maintain tolerance inindividuals.

SUMMARY OF THE INVENTION

[0005] The invention provides for methods and compositions for inducingand maintaining tolerance to epitopes and antigens containing thoseepitopes. The methods and compositions are useful to identify noveltolerogenic epitopes or antigens containing such epitopes. The methodsand composition are also useful for inducing and maintaining toleranceto epitopes or antigens containing the epitopes associated withautoimmune or allergic immune responses.

[0006] The compositions include an expression cassette and a vector. Theexpression cassette and vector can be used to form transformed cells.The expression cassette comprises a DNA sequence coding for a fusionimmunoglobulin operably linked to transcriptional and translationalcontrol regions functional in a hemopoietic or lymphoid cell. The fusionimmunoglobulin has at least one heterologous tolerogenic epitope at theN-terminus variable region of the immunoglobulin molecule. A vectorincludes the expression cassette and is a vector that can provide forstable maintenance, i.e. provide for gene expression of the expressioncassette, in the hemopoietic or lymphoid cell throughout the lifetime ofthe cell. Hemopoietic or lymphoid cells are stably transformed with avector to provide transformed cells expressing the fusionimmunoglobulin.

[0007] The invention also includes pharmaceutical compositions. Apharmaceutical composition comprises an amount of a fusionimmunoglobulin sufficient to induce and/or maintain tolerance combinedwith a pharmaceutically acceptable excipient. The fusion immunoglobulinincludes at least one heterologous tolerogenic epitope at the N-terminusvariable region of the immunoglobulin.

[0008] The invention also provides methods for identifying epitopes orantigens containing epitopes that can serve as novel tolerogens. Themethods involve stably transforming cells with an expression cassettecoding for a fusion immunoglobulin to form a population of transformedcells producing or expressing the fusion immunoglobulin. The fusionimmunoglobulin having one or more than one epitope from an antigensuspected of being capable of inducing tolerance can be screened for theability to induce tolerance to the epitope in a variety of ways. Onemethod of determining whether the fusion immunoglobulin can inducetolerance is to administer a tolerogenic amount of the fusionimmunoglobulin to an animal. In another method, the transformed cellsexpressing the fusion immunoglobulin can be administered to an animal todetermine whether tolerance to the epitope can be induced and/ormaintained. In a third method, epitopes or antigens containing theepitope can be identified by reactivity with allergic or autoimmuneimmune serum or lymphocytes.

[0009] The invention also includes methods of inducing and maintainingtolerance to an epitope in an animal. One of the methods involvesadministering a tolerogenic amount of a fusion immunoglobulin sufficientto induce and/or maintain tolerance to the heterologous epitope on thefusion immunoglobulin. In another method, transformed cells expressing afusion immunoglobulin are administered to an animal to induce andmaintain tolerance. In another method, a pharmaceutical compositionincluding a fusion immunoglobulin is administered to induce tolerance tothe heterologous epitope and transformed cells expressing the fusionimmunoglobulin are then administered to maintain tolerance to theheterologous epitope.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1: Strategy for preparation of a murine DNA construct codingfor a fusion immunoglobulin including the 12-26 epitope of λ-CIrepressor protein at the N-terminus of IgG1: (A) A map of plasmid pSNRcontaining the genomic sequence for a λ1 H chain, modified as describedin Example 1. (B) Restriction map and sequence showing the DNA sequencecoding for the 12-26 epitope as combined with the DNA sequence codingfor the variable region of the heavy chain.

[0011]FIG. 2: Detection of a heterologous epitope on the 12-26-IgGfusion protein. The 12-26-IgG1 construct (Q3), as well as the controlpSNR construct (P6) were electroporated into J558L myeloma cells, whichsynthesize only λ light chains. Recombinant IgG's were purified frombulk supernatants of transformed cells with anti-mouse IgG-sepharose orprotein-A-sepharose columns. Western blotting: samples wereelectrophoresed on 10% SDS-PAGE. Gels were transferred to nitrocelluloseand probed with anti-mouse IgG (left lanes) or with anti-12-26monoclonal antibody B3.11 (right lanes) plus alkalinephosphatase-conjugated antibodies as secondary reagents.

[0012]FIG. 3: ELISA inhibition curves. Pre-titrated monoclonal antibodyB3.11 was mixed with increasing amounts of 12-26 peptide, 12-26 peptidechemically coupled to rabbit gamma globulin (RGG/12-26), or Q3(recombinant fusion protein 12-26 IgG1).

[0013]FIG. 4: Tolerance induction by 12-26-IgG fusion protein asdetermined in vitro. Spleen cells were cultured for 18 hours withincreasing amounts of 12-26 peptide or 12-26-IgG fusion protein (Q3.13)or a 12-26-rabbit gamma globulin (RGG) conjugate. Cells were then washedand challenged with an antigen containing the 12-26 epitope(12-26-fagellin) and ELISA assays were done on day 4 supernatants.

[0014]FIG. 5: In vivo tolerance induction with 12-26-IgG. Balb/c micewere injected with a tolerizing dose of control IgG (P6) at 1 mg/mouse[solid bars], the 12-26 peptide at 100 μg/mouse [open bar], the chemicalconjugate of 12-26 chemically conjugated to rabbit gamma globulin(12-26-RGG) at 1 mg/mouse [stripped bar] and the fusion immunoglobulin(Q3.13) at 1 mg/mouse [dash-dot bar]. After 7 days, spleen cells wereevaluated for responsiveness to in vitro challenge with an antigencontaining the 12-26 epitope as described in FIG. 4.

[0015]FIG. 6A: Western blot showing expression of the 12-26 peptide insupernatants from A20.2J cells infected with MBAE-12-26 vector.Supernatants were slot-blotted on nitro-cellulose and probed withanti-12-26 monoclonal antibody B3.11. MBpepA, MBpepB, MBpepC, and MBpepDrepresent individually infected A20.2J clones producing the 12-26peptide coding for MBAE-12-26-vector.

[0016]FIG. 6B shows proliferation of a T-cell anti-12-26-IgG TH1 clonein response to incubation with supernatants from A20 cells infected withMBAE-12-26 vector or control supernatants.

[0017]FIG. 7 shows construction of an MBAE retroviral vector containingthe DNA sequence coding for the 12-26 epitope.

[0018]FIG. 8 shows a Southern blot of cDNA prepared from reversetranscribed polymerase chain reaction (PCR) products from MBAE-12-26infected bone marrow cells after maturation in irradiated recipients.Peripheral blood cells were obtained from mice 2 weeks after receivinginfected bone marrow cells. RNA was reverse-transcribed and PCRperformed with V_(B) and 12-26 primers. The gels were probed with anoligonucleotide probe complementary to the DNA sequence coding for the12-26 epitope. The experiment demonstrates expression of mRNA coding forthe 12-26 epitope based on RT-PCR of RNA from peripheral blood cells at2 weeks after bone marrow transplantation.

DETAILED DESCRIPTION OF THE INVENTION

[0019] The invention provides for compositions and methods for inducingand maintaining tolerance to antigens. The compositions include anexpression cassette and vector comprising a DNA sequence that codes fora fusion immunoglobulin operably linked to transcriptional andtranslational control regions functional in a hemopoietic cell orlymphoid cell. The fusion immunoglobulin has at least one heterologousepitope located at the N-terminus of the variable region of theimmunoglobulin chain. The vectors are preferably those vectors that canprovide for stable integration of the expression cassette into ahemopoietic cell. The invention also includes cells transformed with thevectors. Fusion immunoglobulins having a heterologous epitope at theN-terminus can be used in a pharmaceutical composition that provides forinduction of tolerance to the epitope and/or its antigen. The inventionalso provides for methods of identifying novel tolerogenic antigens andepitopes, as well as methods for inducing and maintaining tolerance toan antigen.

[0020] As used herein, the term “antigen” refers to an agent that iscapable of eliciting an immune response in an animal.

[0021] An “epitope” is a portion of the antigen that is capable ofeliciting an immune response and combines with an antibody specific forthat portion of the antigen.

[0022] A “heterologous epitope” is an epitope that is not normallyassociated with the immunoglobulin carrier molecule. It is obtained orderived from an antigen that is not the same as the immunoglobulincarrier molecule.

[0023] A “hemopoietic cell” is a cell that can form blood cells includelymphocytes and macrophages from such tissues as bone marrow cells andother extramedullary tissues.

[0024] An “expression cassette or vector” is stably maintained in ahemopoietic or other cell type when it is either integrated into thechromosome so that the expression cassette or vector is replicated andtransmitted to progeny cells or is maintained in the cell without lossof functional activity, i.e. gene expression, over the lifetime of thecell.

[0025] A “tolerogenic epitope” is an epitope that can induceimmunological unresponsiveness to the epitope and/or an antigencontaining an epitope. A tolerogenic epitope is selected because of adesire to induce immunological unresponsiveness to the epitope and/or anantigen containing the epitope. A tolerogenic epitope can be identifiedas an epitope that can stimulate an immune response if appropriatelypresented to the immune system or it can be an auto- or self-antigenwhich may not normally elicit an immune response. A tolerogenic epitopecan interact with T cells or B cells or both. Suitable tolerogenicepitopes that can be selected for are preferably those epitopes and/orantigens associated with autoimmune disease or allergic reactions.

[0026] A. Expression Cassettes and Vectors

[0027] An expression cassette of the invention includes a DNA sequenceencoding a fusion immunoglobulin operably linked to transcriptional andtranslational control regions functional in a hemopoietic or lymphoidcell. The fusion immunoglobulin includes at least one heterologoustolerogenic epitope at the N-terminus variable region. The expressioncassette is preferably incorporated into a vector that provides forstable maintenance and expression of the expression cassette in the hostcell. If the host cell is a hemopoietic cell, the vector is preferably avector that provides for integration of the vector into the chromosomeof the hemopoietic cell. If the host cell is a lymphoid cell line, thevector can be a non-integrated vector such as a plasmid as long as itprovides for stable maintenance and expression of the expressioncassette over the lifetime of the cell. The expression cassettes andvectors of the invention are useful to provide fusion immunoglobulins touse as tolerogenic agents and/or to provide for maintenance of toleranceto an antigen and/or epitope.

[0028] A DNA sequence encoding a fusion immunoglobulin can be obtainedand constructed using standard methods as described in Current Protocolsin Molecular Biology, Chapter 3, J. Wiley/Greene Press (1992). DNAsequences encoding immunoglobulins can be obtained using known methodssuch as described by Hebell et al., Science, 254:102 (1991) and Huse etal., Science, 246:1275 (1989). Briefly, heavy and light chain sequencescan be obtained by using reverse transcriptase polymerase chain reaction(RT-PCR) of messenger RNA (mRNA) isolated from spleen cells or,preferably, hybridomas producing an antibody of known specificity. Theprimers can be designed to amplify the variable light and heavy chainsequences including the Fd fragment (V_(B)-CH1). Examples of suchprimers are disclosed in Huse et al, cited supra. and Ballard et al.,PNAS, 83:9626 (1986). Typically such primers are designed to includerestriction enzyme recognition sequences at both ends of the sequence tobe amplified. The restriction endonuclease recognition sequences areknown to those of skill in the art and can be selected to provide forease of cloning into a vector at a specific location.

[0029] The DNA sequences encoding the immunoglobulin's light and heavychains are preferably cDNA sequences so that any intervening sequenceDNA has been removed and a fully functional immunoglobulin is encoded bythe DNA sequence. The DNA sequence encoding the immunoglobulin moleculecan encode a complete immunoglobulin having both heavy and light chainswith the Fc fragment or it can encode portions of the immunoglobulinsuch as Fab fragment, F(ab)₂ fragment, or just the heavy chain.Modifications to the DNA sequence coding for the heavy chain can be madeand still result in a fusion immunoglobulin molecule when the DNAsequence coding for the heavy chain is expressed in a cell of B celllineage that can supply light chains to form the immunoglobulin. The DNAsequence can code for a secreted or membrane form of the immunoglobulinmolecule.

[0030] Suitable examples of a DNA sequence coding for the heavy chain ofan antibody specific for nitrophenyl are described by Hebell et al.,cited supra. IgG1 or IgG2 (mouse) are preferred as carrier molecules forinducing tolerance. The DNA sequence preferably codes for the heavychain of IgG1 or IgG2 types of immunoglobulin.

[0031] A DNA sequence coding for at least one tolerogenic epitope of anantigen can be obtained and prepared by standard methods. If the epitopeis a small peptide of 15-20 amino acids, the nucleotide sequenceencoding that epitope can be synthesized using automated DNA synthesis.If the DNA sequence codes for all or a portion of an antigen (i.e.,codes for multiple epitopes), the DNA sequence coding for that antigencan be isolated and subcloned using published methods. The DNA sequencescoding for all or a portion of some antigens can also be identified bysearching in a database such as GenBank. Once the sequence is identifiedin such a database or by reference to publications, the DNA sequencecoding for all or a portion of an antigen can be obtained by automatedsynthesis or by polymerase chain reaction (PCR). For example, the DNAsequence coding for antigen E of ragweed pollen has been disclosed byRafner et al., J. Biol. Chem., 266:1229 (1991); and Kuo et al.,Molecular Immunol., 30:1077 (1993). Epitopes of antigen E have also beenidentified as described by Olson, J. Immunol., 136:2109 (1986); and Bondet al., J. Immunol., 146:3380 (1991). A DNA sequence encoding one ormore of the epitopes of antigen E can be obtained by standard methods asdescribed in Kuo et al., cited supra.

[0032] Suitable antigens are those that it would be desirable to induceand maintain immunological unresponsiveness to the epitope and/orantigen containing the epitope. Such antigens include pollen, ragweed,dustmites, and other known allergens. Suitable antigens also includeautoantigens such as clotting factor VIII, acetylcholine receptors,collagen, myelin basic protein, thyroglobulin, and histocompatibilityantigens. A suitable antigen also includes the epitopes from the λ-CIrepressor protein. The amino acid sequences of many of these antigens aswell as epitopes of these antigens are known to those of skill in theart. The preferred antigens include antigen E of ragweed and clottingfactor VIII. The DNA sequences encoding suitable antigens can beobtained and prepared as described herein and in accord with publishedmethods.

[0033] Before a DNA sequence coding for at least one tolerogenic epitopeof an antigen is obtained and prepared, the epitope and/or antigen isselected. The epitope and/or antigen can be a single epitope or it canbe all or a portion of an antigen containing many epitopes. The epitopecan be one that interacts with T cells, or one that interacts with Bcells, or one that interacts with both T and/or B cells.

[0034] The selection of the epitope and/or epitopes can be made based onthe following criteria. Epitopes are first selected for the ability toinduce tolerance to the peptide or an antigen containing the epitope,preferably an antigen associated with an allergic response or autoimmuneresponse. Secondly, if tolerance is desired to a large and complexantigen, more than one epitope can be selected to be combined into afusion immunoglobulin. Preferably, the entire antigen may be included inthe fusion immunoglobulin. Thirdly, epitopes may be selected if B and/orT cell tolerance is desired. Certain epitopes are known to those ofskill in the art to be recognized by T cells and not B cells and viceversa. Fourthly, epitopes can be selected on the basis of reactivitywith immune serum or lymphocytes from individuals having an allergic orautoimmune response to an antigen. For example, an epitope known to beimmunodominant or to stimulate a strong autoantibody response can beselected so that the portion of the antigen included in the fusionimmunoglobulin includes that epitope. Fifthly, if there is little or noinformation known about epitopes on the antigen, it may be desireable toinclude the entire antigen in the fusion immunoglobulin.

[0035] The DNA sequence coding for an epitope can include an epitope ofabout 5-6 amino acids or an antigen having a molecular weight of up toabout 100,000 daltons. The preferred size range is about 9 amino acidsto about 50,000 daltons. For example, epitopes recognized by T cellshave a consensus sequence including about 9 amino acids. It is believedthat the minimal size of an epitope is about 5-6 amino acids. Themaximum size of the antigen that can be presented in a fusion protein isthe size that allows for the folding of both the antigen and theimmunoglobulin carrier molecule. A preferred antigen is the A2 fragmentof clotting factor VIII that has a molecular weight of about 40,000daltons.

[0036] Once the epitope is selected and the DNA sequence encoding thatepitope is obtained, the DNA sequence coding for the epitope is combinedwith the DNA sequence coding for the immunoglobulin to form a DNAsequence coding for a fusion immunoglobulin. The DNA sequence coding forthe epitope is preferably combined with a DNA sequence for theimmunoglobulin at the N-terminal variable region of the heavy chain inframe and in proper orientation. The location of the combination of theDNA sequence coding for the epitope can vary depending on the desiredlocation of the epitope in the fusion immunoglobulin. If the epitope isthe entire antigen or a large portion of the antigen (i.e., having amolecular weight of about 25,000 to about 100,000 daltons), the locationof the epitope on the fusion immunoglobulin id such that it would allowfolding of both the immunoglobulin carrier molecule as well as theantigen or the portion of the antigen. When the antigen and/or portionof the antigen is an epitope, it is preferably fused with theimmunoglobulin at the amino terminus of the heavy chain at the aminoacids at the N-terminus first framework region. Smaller epitopes (i.e.,those containing about 5-50 amino acids) can be located at the firstN-terminal framework region or within other regions on the variableportion of the immunoglobulin chain as long as the epitope remainsexposed on the outer surface of the immunoglobulin molecule. Preferably,small epitopes can also be combined with the immunoglobulin at the aminoacids of the first N-terminal framework region of the heavy chain.

[0037] Optionally, the DNA sequence coding for at least one heterologoustolerogenic epitope can include flanking DNA sequences on one or bothends of the DNA sequence. These flanking DNA sequences can includerestriction endonuclease recognition sequences and/or can include a DNAsequence encoding a portion of the immunoglobulin sequence at thelocation where the two DNA sequences are to be combined. For example, aDNA sequence coding for an epitope that is combined at the firstN-terminus framework region of a heavy chain of an immunoglobulinmolecule can include a flanking DNA sequence encoding the first 5 aminoacids of the first framework region on either or both ends of the DNAsequence coding for the epitope. The flanking DNA sequence can alsoinclude a recognition sequence for a restriction enzyme. The flankingDNA sequence is preferably about 3 to about 21 nucleotides long. Whenthe flanking DNA sequence encodes a portion of the immunoglobulin aminoacid sequence, that sequence is selected at the location of the point ofcombination of the epitopal DNA sequence with the immunoglobulinsequence. The flanking DNA sequence coding for a portion of theimmunoglobulin amino acids can provide for amino acids in the fusionimmunoglobulin that assist in the proper folding of both the epitopeand/or antigen and the immunoglobulin at the point of fusion. Theflanking DNA sequence can also insure that the DNA sequence coding forthe epitope are combined with the DNA sequence coding for theimmunoglobulin in frame and in proper orientation.

[0038] The DNA sequences coding for the immunoglobulin and the epitopeare combined using standard subcloning methods. The combination of thetwo DNA sequences can be assisted by forming the DNA sequence encodingthe epitope with flanking DNA sequences having certain restrictionenzyme recognition sequences. These flanking sequences provide one ofskill in the art with the ability to select the location at which theDNA sequence coding for the epitope will be combined with the DNAsequence coding for the fusion immunoglobulin and to insure thesequences are combined in frame and in proper orientation. When the DNAsequences coding for the immunoglobulin and the epitope are combined,they form a DNA sequence coding for a fusion immunoglobulin or a fusionheavy chain of an immunoglobulin molecule.

[0039] It should be understood that, due to the degeneracy of thegenetic code, there are a number of DNA sequences that can code for animmunoglobulin and an epitope that have the same amino acid sequence.This set of sequences is a finite set and can be determined based on theamino acid sequence of the epitope and immunoglobulin. Alternative DNAsequences that code for an immunoglobulin molecule and an epitope withthe same amino acid sequence are contemplated by and included within thescope of the invention.

[0040] The DNA sequence coding for a fusion immunoglobulin can then becombined with transcriptional and translational control regionsfunctional in a hemopoietic or lymphoid cell. A control region that isimportant for expression of the DNA sequence coding for a fusionimmunoglobulin includes a promoter. A suitable promoter is one that canfunction in a hemopoietic or lymphoid cell. The promoter preferablyprovides for constitutive expression of the DNA sequences coding for thefusion immunoglobulin. The promoter also preferably provides for anamount of the fusion immunoglobulin to induce and/or maintain tolerance.Suitable examples of promoters include the β-actin promoter, the SV40promoter, and the LTR Rous sarcoma virus promoter.

[0041] Other transcriptional and translational control regions includeenhancer sequences and transcription termination and polyadenylationsequences. Enhancer sequences can be combined with and are usually foundwithin or adjacent to promoter sequences. Certain enhancer sequences,such as those from SV40, are active in many mammalian cells and providefor stimulation of transcription up to 1,000-fold from the homologous orheterologous promoters. Polyadenylation sequences are found downstreamfrom the coding sequence and provide for proper formation of mRNA.Polyadenylation sequences can be obtained from SV40. Transcriptiontermination sequences are found downstream from the polyadenylationsequences within a few hundred nucleotides.

[0042] These transcriptional and translational control regions areavailable in commercially available vectors. A DNA sequence encoding afusion immunoglobulin or fusion heavy chain can be combined withtranscriptional and translational control regions in frame and in properorientation by subcloning into a vector having these control regions toform an expression cassette.

[0043] Vectors can be selected for the ability to provide for stablemaintenance and/or gene expression in a hemopoietic or lymphoid cell. Avector is stably maintained in a cell if it can provide for expressionof a fusion immunoglobulin over the lifetime of the cell. Stablemaintenance can include maintenance and expression of a plasmid in aeukaryotic cell, preferably a cell such as a lymphoid cell. In thatcase, the plasmid including an expression cassette is not autonomouslyreplicated or does not become integrated into the chromosome. Thelifetime of a cell, such as a lymphoid cell, is about 14 to 60 days inthe mouse or can be several years in humans. A plasmid vector containingan expression cassette can also be maintained in a lymphoid cell linesuch as the J558L cells without being replicated.

[0044] A vector can also be selected to provide for integration of theexpression cassette into the chromosome of the host cell, such as ahemopoietic cell. In a hemopoietic cell from the bone marrow of ananimal, the vector is introduced into a mixed population of cells, someof which are dividing cells and some of which have not yet begundividing. The vector can integrate into the chromosome and then bereplicated along with the chromosome and transferred to progeny cells.The vector is stably integrated if gene expression can be detected inthe cell population at about 1 to 12 weeks after infected cells areintroduced into an animal or cultured in vitro.

[0045] Suitable vectors include the plasmids such as PSNR1, pEMBL,pBR322, pRSA101, pUC118, pUC119, pBluescript, and pComb (Barbas et al.,PNAS, 88:7978 (1991)). Suitable vectors also include viral vectors suchas baculovirus and retroviral vectors such as the MBAE vector (Chamberset al., PNAS, 89:1026 (1992)). The preferred vector for hemopoieticcells is the MBAE vector.

[0046] A bacterial strain containing a plasmid vector having a DNAsequence that codes for fusion heavy chain has been designated E. coliDH5α (pQ3.EZ). The bacterial strain carries the plasmid pQ3.EZ whichcodes for fusion heavy chain that has a 12-26 amino acid epitope fromλ-C1 repressor protein combined at the N-terminus first framework regionof the heavy chain of an antibody specific for nitrophenyl. Thebacterial strain has been deposited with the American Type CultureCollection at Rockville, Md. on ______, 1994 and given Accession No.______.

[0047] In a preferred version, a DNA sequence coding for an epitope suchas the 12-26 epitope from the λ-C1 repressor protein is combined withthe DNA sequence coding for an immunoglobulin variable region at thefirst N-terminal framework region of the heavy chain to form a DNAsequence coding for a fusion heavy chain. The DNA sequence coding for afusion heavy chain is combined with a β-actin promoter in an MBAEretroviral vector. The vector is preferably used to transform bonemarrow cells or other B cell lineage cells that can produce lightchains. The light chains combine with the fusion heavy chain to form afusion immunoglobulin. Alternatively, a DNA sequence coding for a lightchain could be included in the same vector as that coding for the fusionheavy chain to provide for expression of a fusion immunoglobulin.

[0048] B. Transformed Cells

[0049] Vectors containing expression cassettes coding for a fusionimmunoglobulin are used to transform cells. The transformed cells areused in methods of identifying novel tolerogenic epitopes and to producea fusion immunoglobulin. Transformed cells can also be introduced intoanimals for induction and maintenance of tolerance to the heterologousepitope expressed by the transformed cells or to an antigen containingthe heterologous epitope.

[0050] Suitable cells for transformation include hemopoietic cells,lymphoid cells, and lymphoid cell lines. The cells include bone marrowcells, lymphoid cells, and the J558L lymphoid cells. Host cells arepreferably those that are capable of forming and secretingimmunoglobulin molecules. The cell population transformed preferablyincludes cells of B cell lineage and are those that synthesize lightchains endogenously. Transformed cells that will be administered toanimals are preferably syngeneic or share identical histocompatibilityantigens to avoid rejection of the injected cells. For screening assays,bacterial host cells such as E. coli and the like can be suitable.

[0051] The vector can be introduced into cells using a variety ofmethods known to those of skill in the art such as calcium phosphatemediated transfection, polybrene mediated transfection, protoplastfusion, electroporation, and lipsomal mediated transfection.

[0052] Once the expression cassette is introduced into the cells, thetransfected cells can be initially selected by detecting the presence ofa selectable marker gene present on the vector. If the transfected cellsare bone marrow cells or lymphoid cells, no selection may be employed.Transfected cells can then be screened for the presence and/orexpression of the expression cassette coding for a fusionimmunoglobulin. Transfected cells can be screened for the presence of anexpression cassette using one or more techniques such as Southern blot,Northern blot, reverse transcriptase PCR, Western blot, ELISA, andimmunofluorescence. Detectably labelled DNA probes can be used inSouthern and/or Northern blots. The probes are sufficientlycomplementary to nucleotide sequences coding for the epitope or aportion of an antigen or an antigen so that the probe of about 50 to 100nucleotides hybridizes under high stringency conditions. Primers forreverse transcriptase PCR can be designed as described previously toamplify cDNA sequences coding for the variable heavy and light chains ofthe immunoglobulin molecule.

[0053] Transfected cells in which the fusion immunoglobulin is beingexpressed can also be detected using a Western blot, ELISA orimmunofluorescence. Amounts of fusion immunoglobulins being expressedcan be detected using quantitative Western blot.

[0054] The amount of fusion immunoglobulin produced in a particular hostcell type and with a particular promoter/enhancer sequence can beevaluated using a quantitative Western blot. The promoter/enhancersequences providing for the most amount of constitutive expression ofthe fusion immunoglobulin can be determined by comparing the amount offusion immunoglobulin produced in the same type of host cell over thesame amount of time. A promoter/enhancer can be selected that wouldprovide for a sufficient amount of fusion immunoglobulin to induceand/or maintain tolerance. The amount of a fusion immunoglobulin thatwill induce tolerance can vary in accordance with factors describedherein and can be determined using standard methods.

[0055] C. Pharmaceutical Compositions

[0056] The invention also provides pharmaceutical compositions includinga tolerogenic amount of a fusion immunoglobulin in a pharmaceuticallyacceptable excipient. The fusion immunoglobulin has at least oneheterologous tolerogenic epitope on the N-terminal variable region ofthe immunoglobulin. Preferably, the heterologous tolerogenic epitope iscombined with the immunoglobulin adjacent to the first N-terminalframework region of the heavy chain. The fusion immunoglobulin iscombined with a pharmaceutically acceptable excipient in amountseffective to induce tolerance to the tolerogenic epitope or to anantigen containing the epitope in an animal. The pharmaceuticalcomposition can be administered to an animal to induce and/or maintaintolerance to the tolerogenic epitope. Induction of tolerance to theepitope or epitopes can minimize animal allergic reactions or thesymptoms of autoimmune disease.

[0057] Fusion immunoglobulins can be isolated from transformed cellsusing standard methods. Fusion immunoglobulins can be isolated from cellsupernatants by passage through protein A or other affinity columns inaccord with standard methods.

[0058] Suitable tolerogenic epitopes are those epitopes associated withallergic or autoimmune responses. A tolerogenic epitope is one that canbe administered in such a way as to result in immunologicalunresponsiveness to the epitope and/or an antigen containing theepitope. If the epitope is one that stimulates an immunodominantresponse, tolerance to that epitope can also result in tolerance to anantigen containing the epitope. Specific examples include antigen E orantigen K of ragweed pollen, dust mite antigens, heterologoushistocompatibility antigens, clotting factor VIII, acetylcholinereceptors, myelin basic protein, and thyroglobulin. The fusionimmunoglobulin can contain a single tolerogenic epitope or a multipletolerogenic epitopes. Preferably, the tolerogenic epitope is an epitopethat is immunodominant in the allergic or autoimmune response.

[0059] The amount of the fusion immunoglobulin effective to inducetolerance in an animal depends on a factors but can be readilydetermined by one of skill in the art using standard dose responsemethods. The factors include the size of the animal to be treated, thenumber and type of epitopes, the type of tolerance, the age of theanimal, the route and number of times of administration, and theduration of the tolerance desired.

[0060] The age of the animal can be an important factor in determiningthe effective tolerogenic amount of an epitope. A neonatal or infantanimal may require about 100 to 1000-fold less of a single dose of afusion immunoglobulin administered intravenously than that required byan adult of the fusion immunoglobulin in order to induce tolerance tothe epitope.

[0061] A tolerogenic amount of a fusion immunoglobulin also depends onthe size of the animal and is typically about 10 to 100-fold higher (forB-cell tolerance) than the amount of the antigen and/or epitope given tothe animal to elicit a protective immune response, except in the case oflow dose tolerance. A tolerogenic amount of an antigen per unit of massis typically about 1 to 40 mg/kg of body weight to induce high dosetolerance for an epitope or antigen administered as a single doseintravenously to an animal. Low dose tolerance is also observed in somecases and can be obtained after multiple (>4) doses of submicrogramquantities in saline at weekly intervals intraperitoneally orintravenously.

[0062] Another factor that can vary the tolerogenic amount of a fusionimmunoglobulin is whether the fusion immunoglobulin includes more thanone epitope and whether those epitopes are immunodominant. If the fusionimmunoglobulin has multiple epitopes, some of which are immunodominant,about a 10-fold lower dose of fusion immunoglobulin can induce tolerancewhen administered as a single dose to an animal intravenously.

[0063] The tolerogenic amount of a fusion immunoglobulin can also varydepending on whether a T cell or B cell tolerance is desired. Typically,T cell tolerance requires a dose of antigen or epitope about 10 to100-fold less than for B cell tolerance to that same epitope or antigen.

[0064] Another factor is the persistence of the fusion immunoglobulin inthe animal's circulation. A more slowly metabolized antigen provides formaintenance of tolerance for longer periods of time, typically about 2to 10-fold greater time of maintenance of tolerance. The catabolic rateof epitopes or antigens depends on the half-life of isologous or theheterologous carrier immunoglobulin as well as the nature of the epitopeor epitopes. The half-life rate of isologous or heterologousimmunoglobulin is about 7 to 21 days (mouse). Epitopes having modifiedor unusual amino acids, such as D amino acids as well as complexantigens or epitopes, may not be degraded as rapidly as other types ofepitopes.

[0065] Mode of administration can also influence the tolerogenic amountof the fusion immunoglobulin necessary. In the usual case, intravenousadministration is the preferred route for inducing tolerance. The numberof times the antigen is administered can also influence the amount offusion immunoglobulin required per administration.

[0066] An effective tolerogenic amount for a particular heterologoustolerogenic epitope on a fusion immunoglobulin can be determined byconducting in vivo or in vitro dose response assays. The in vitro doseresponse assays can be conducted, for example, by using standardlymphocyte proliferation assays. For example, lymphocytes from anallergic or autoimmune animal can be combined with different doses ofthe fusion immunoglobulin and proliferation measured.

[0067] In vivo dose response can be determined by administeringdifferent doses of the fusion immunoglobulin in an excipient to ananimal. The lack of immune responsiveness to the heterologoustolerogenic epitope can be determined by measuring the specific antibodyresponse to the heterologous tolerogenic epitope or lymphocyteproliferation to a challenge dose of the fusion immunoglobulin.

[0068] Induction of tolerance is evaluated by measuring a decrease inimmunological unresponsiveness. Methods of measuring immunologicalresponsiveness can be conducted with in vivo or in vitro antigenpresentation and challenge and are known to those of skill in the art.For example, the amount of antibody specific to the epitope and/orantigen can be measured as well as lymphocyte proliferation in responseto a challenge with the epitope or fusion immunoglobulin. The decreasein immunological Identification of novel tolerogenic epitopes could beuseful in diagnosis and treatment of autoimmune and allergic immuneresponses. One method includes the steps of providing a vector includinga DNA sequence coding for a fusion immunoglobulin operably linked totranscriptional and translational control regions functional in a hostcell. The fusion immunoglobulin has at least one heterologous epitope atthe N-terminus variable region. The epitope can be one that is suspectedof being able to induce tolerance. Cells are stably transformed with thevector as described previously. Transformed cells expressing the fusionimmunoglobulin or the isolated fusion immunoglobulin are analyzed forthe ability to immunoreact with immune serum or lymphocytes fromallergic or autoimmune animals. Tolerance induction to a fusionimmunoglobulin identified by reactivity with immune serum or lymphocytesfor autoimmune or allergic animals can be evaluated by in vitro or invivo methods known to those of skill in the art. For example, fusionimmunoglobulins that react with immune serum and/or stimulate lymphocyteproliferation can be administered to an animal and induction andmaintenance of tolerance can be assessed as described herein.

[0069] In another method, the transformed hemopoietic or lymphoid cellscan be introduced into an animal and induction and maintenance oftolerance to the heterologous epitope can be determined using assays forevaluating specific immunological responsiveness to the epitope asdescribed previously.

[0070] Some epitopes and antigens are known to elicit immune responses.Some epitopes and antigens are known to elicit immunodominant immuneresponses associated with allergic or autoimmune immune responses. Thoseepitopes that elicit immune responses may or may not induce tolerancewhen presented in a fusion immunoglobulin. Epitopes of some antigensknown to be associated with allergic or autoimmune immune responses havenot been identified. The methods of the invention can be utilized todetermine whether an epitope known to elicit an immune response caninduce tolerance when presented in a fusion immunoglobulin or toidentify novel tolerogenic epitopes of antigens.

[0071] In one method, a vector comprising a DNA sequence coding for afusion immunoglobulin operably linked to transcriptional andtranslational control regions functional in the hemopoietic or lymphoidcell is transformed into a hemopoietic or lymphoid cell. The fusionimmunoglobulin can include an epitope known to elicit an immune responseor a novel tolerogenic epitope. The promoter/enhancer sequencespreferably provide for expression of the fusion immunoglobulin in ahemopoietic or lymphoid cell at a level sufficient to induce toleranceto the epitope in vivo or in vitro. Such a promoter can be identifiedand screened for in an in vitro assay as described herein. The amount offusion immunoglobulin that can induce tolerance in animals can bedetermined using standard dose response methodology.

[0072] The transformed cells are introduced into an animal. Whentransformed hemopoietic cells are introduced into an animal, preferablythe animal has been irradiated before introduction of the transformedcells to destroy endogenous hemopoietic cells. The transformed cells areadministered to an animal by intraperitoneal or intravenous injection.The animals are then analyzed for induction of tolerance to the epitopeafter about 2 to 20 days. Tolerance can be detected by measuring thespecific antibody response or lymphocyte proliferation response to theheterologous tolerogenic epitope. A decrease in the specific antibody orlymphocyte proliferative response to the epitope of about 2 to 100-fold,preferably 10- to 100-fold, indicates tolerance to the epitope.

[0073] Preferably, the screening assays for identifying tolerogenicepitopes are conducted in mice. The transformed cells can be syngeneicmouse cells derived from another genetically identical mouse, or can behuman hemopoietic or lymphoid cells. For example, screening assays canbe done using human bone marrow tissue transformed with a vector. Thehuman bone marrow tissue is then administered to immunodeficient micesuch as the SCID-SCID mice according to the method described by Chamberset al., cited supra. Tolerance can be evaluated in the SCID-SCID mice byexamining either the specific antibody response to the epitope or thelymphocyte proliferation response.

[0074] Another method of the invention provides for screening for noveltolerogens, preferably those associated with autoimmune or allergicimmune responses. In this method, epitopes of antigens associated withallergic or autoimmune responses are screened for the ability toimmunoreact with immune serum or to stimulate lymphocyte proliferationfrom animals having an allergic or autoimmune response. For example,different cDNA sequences coding for portions of a complex antigen suchas clotting factor VIII can be combined with a DNA sequence coding forN-terminus variable region of an antibody to form a library of cDNAsequences coding for fusion immunoglobulins with different epitopesderived from clotting factor VIII. The DNA sequences coding for epitopescan be generated randomly, or can be selected to encode overlappinglinear amino acid sequence, or can be selected based upon the likelihoodthat the amino acids encoded by the DNA sequence are exposed (based ontertiary structure) on the surface of the clotting factor VIII molecule.The cDNA sequences coding for different portions of the antigen can becombined with cDNA sequences for the N-terminus variable region of animmunoglobulin, preferably at the first N-terminus framework region ofthe heavy chain as described previously.

[0075] A phagemid vector system such as pComb can be used to generate acDNA library of heavy and light chains of antibodies having cDNAsequences coding for different portions of an antigen combined asdescribed above. The phagemid vector can be constructed to carry thesecDNA sequences using standard restriction enzyme digestion and ligationmethods as described in Barbas et al., PNAS, 88:7978 (1991). Thephagemid library can be screened for immunoreactivity with immune serumfrom allergic or autoimmune animals in a panning and/or filter Westernblot assay similar to those described by Barbas et al., cited supra.

[0076] Briefly, the phagemid vectors carrying the Fab fragments with atleast one heterologous epitope derived from an antigen are transformedinto a E. coli strain. The E. coli strain is grown in the presence ofantibiotics to select for those strains carrying the phagemid vector.Phage can be isolated and then screened for binding to wells coated withimmune serum from an allergic or autoimmune animal as described byBarbas et al., cited supra. Adherent phage are eluted using elutionbuffer. Eluted phage can be transferred into E. coli cells and coloniescan be examined for the presence of a phagemid carrying a Fab fragmentwith a heterologous epitope using a filter Western blot type assay withimmune serum from an allergic or autoimmune animal.

[0077] Phagemid DNA from positive clones can be isolated and the DNAsequence coding for the fusion Fab can be subcloned into a vector thatcan be used to transform hemopoietic or lymphoid cells. The vector cancontain additional DNA sequences so that a fusion immunoglobulin ratherthan Fab fragment is produced by the transformed cells. The fusionimmunoglobulin having a heterologous epitope that reacts with immuneserum from allergic or autoimmune animals from a positive cloneidentified as described can be isolated and tested for the ability toinduce tolerance in vitro or in vivo. Alternatively, transformed cellscarrying such a vector can be introduced into an animal and induction oftolerance in vivo can be determined as described herein.

[0078] Once novel epitopes and/or fusion immunoglobulins that can inducetolerance are identified, they can be used in pharmaceuticalcompositions and methods for tolerizing animals to the epitopes.Alternatively, the identification of novel tolerogenic epitopesassociated with autoimmune or allergic immune responses could be used instandard diagnostic assays to assess the presence of autoimmune orallergic immune responses or to monitor the effectiveness of treatment.

[0079] E. Methods of Tolerizing an Animal to an Epitope

[0080] The invention also provides methods for inducing and maintainingtolerance to an epitope in an animal. In one method, a pharmaceuticalcomposition including a fusion immunoglobulin is administered to ananimal as described previously. In another method, tolerance can beinduced and maintained in an animal by introducing transformedhemopoietic or lymphoid cells producing the fusion immunoglobulin intothe animal. Without limiting the invention in any way, it is believedthat the persistent production of fusion immunoglobulin carrying theheterologous epitope by the transformed cells in vivo can allow formaintenance of tolerance as well or better than using a pharmaceuticalcomposition of the fusion immunoglobulin.

[0081] In one method, a vector coding for a fusion immunoglobulin thatcan be stably maintained in a hemopoietic or lymphoid cell is provided.The fusion immunoglobulin has at least one heterologous tolerogenicepitope. Hemopoietic or lymphoid cells, such as peripheral blood cells,are transformed with a vector such as MBAE using polybrene. Transformedcells are not typically selected and the entire population ofhemopoietic or lymphoid cells are administered to the animal.Transformed cells can be evaluated for production of a fusionimmunoglobulin in vivo or in vitro by detecting the presence of fusionimmunoglobulin using antibodies or by detecting expression of fusionimmunoglobulin mRNA using RT-PCR or Northern blots. Preferably, thetransformed cell population is analyzed in vitro for production offusion immunoglobulin at a level sufficient to induce and maintaintolerance to the heterologous epitope in an animal.

[0082] Transformed cell population prepared so that the fusionimmunoglobulin is produced at a level sufficient to induce and/ormaintain tolerance are introduced into an animal. The amount of cellsintroduced into the animal is that amount that provides for productionof a fusion immunoglobulin at a level sufficient to induce tolerance,and preferably to maintain tolerance. The animal is monitored forinduction and persistence of tolerance to the heterolugous epitope usingassays as described previously. In some cases, the animals areirradiated sufficiently to destroy endogenous hemopoietic or lymphoidcells before introduction of the transformed cell populations. An animalis considered tolerant to the epitope if about a 2- to 100-fold decreasein immunological responsiveness, such as lymphocyte proliferation orantibody response, is seen. Tolerance is considered to be maintained ifthe tolerant state is maintained at least as long as the tolerant stateinduced with a single intravenous injection of a tolerogenicpharmaceutical composition. In mice, a single injection of a tolerogenicamount of a fusion immunoglobulin can result in tolerance of about 2 to20 days and as long as about 2 months to 6 months. Tolerance could bemaintained throughout the lifetime of the animal.

[0083] Suitable transformed cells include bone marrow cells and lymphoidcells from mice or humans. Suitable animals include inbred strains ofmice including immunodeficient mice such as the SCID-SCID mice.Induction and maintenance of tolerance to epitopes using humantransformed cells can be evaluated by the development of tolerance toepitopes in human transformed cell populations administered to SCID-SCIDmice. Other transformed animal cells, such as bovine transformed cells,can also be evaluated for the induction of tolerance in SCID-SCID mice.

[0084] In another method, a tolerogenic amount of a fusionimmunoglobulin can be used to induce tolerance and tolerance can bemaintained by administration of transformed hemopoietic or lymphoidcells expressing the same fusion immunoglobulin. In the method, atolerogenic amount of a fusion immunoglobulin can be administered as asingle dose as described herein. After a state of immunologicalunresponsiveness is obtained, transformed hemopoietic or lymphoid cellsexpressing the fusion immunoglobulin can be administered to the animal.While not meant to limit the invention, it is believed that thetransformed hemopoietic or lymphoid cells will result in the maintenanceof tolerance to the epitope. The amount of fusion immunoglobulin thatneeds to be expressed when transformed cells are used to maintain ratherthan induce tolerance can be less than that required of cells that bothinduce and maintain tolerance. Typically, administration of about 10 to100-fold less of the fusion immunoglobulin or antigen is required tomaintain rather than induce tolerance.

EXAMPLE I Preparation of Fusion Immunoglobulin p12-26 RecombinantConstructs

[0085] Tolerance to the epitope comprising residues 12-26 of thebacteriophage λ cI protein was studied because this epitope can berecognized by both T and B cells, and it is the major immunodominantepitope of this protein in H-2^(d) mice. This epitope was expressed in afusion protein of mouse IgG having the, epitope at the N-terminus.Isologous IgG1 was chosen for the fusion protein because it is known tobe a tolerogenic carrier. Isologous immunoglobulins (especially IgG's)are likely to make efficient tolerogenic carriers because of theirability to crosslink B-cell Fc receptors and to persist in thecirculation, as well as their lack of “intrinsic immunogenicity”, thatis, the lack of the potential to elicit an immune response in a solubleform. DNA constructs coding for a fusion polypeptide of immunoglobulinIgG containing the 12-26 epitope of λ cI repressor protein were obtainedby modifying plasmid pSNR-1. (See FIG. 1.)

[0086] The major immunodominant peptide of the λ cI repressor protein(residues 1-102) is found at residues 12-26, as described in Nature,343:381 (1990). The DNA sequence coding for this peptide fragment wassynthesized by standard automated methods. The synthetic oligonucleotidefragment coding for the 12-26 epitope has the following sequence (SEQ IDNO:1): 5′ CTG GAG GAC GCG CGG CGG CTG AAG GCG ATA TAC GAG AAG AAG AAG 3′3′ GAC CTC CTG CGC GCC GCC GAC TTC CGC TAT ATG CTC TTC TTC CCT 5′

[0087] The corresponding amino acid sequence encoded by this fragmentis: Leu-Glu-Asp-Ala-Arg-Arg-Leu-Lys-Ala-Ile-Tyr-Glu-Lys-Lys-Lys (SEQ IDNO:2)

[0088] Plasmid pSNR-1 is a plasmid that includes a DNA sequence codingfor the variable heavy chain domain (VH) and heavy chain constantregions 1-3 (CH1-3) from a murine immunoglobulin specific for4-hydroxy-3-nitrophenyl. Plasmid pSNR-1 was constructed as described byBallard et al., PNAS, 83:9626 (1986). The pSNR-1 plasmid was obtainedfrom Douglas Fearon (Johns Hopkins, Baltimore, Md.). To introduce theDNA sequence coding for the 12-26 epitope into the N-terminus of thevariable heavy chain, the plasmid PSNR was manipulated as describedbelow. A 1.3 kbp region of the pSNR-1 plasmid including the codingsequence for VH, 118 bp of DNA sequence 5′ upstream promoter element tothe VH coding sequence coding for a promoter element, and 3′ downstreamintron and IgH enhancer sequences was subcloned using standard methods.This sequence is defined between restriction enzyme sites BamHI andEcoRI, and was subcloned into the plasmid pBS (Stratagene) using BamHIand EcoRI restriction endonucleases. The pBS/VH was digested with PstIunder conditions to isolate a single cut PstI partial digest fragment,as described in Current Protocols in Molecular Biology, cited supra.

[0089] The 12-26 epitope was modified and then inserted into the VHregion of the immunoglobulin at a location that provided for properfolding of that region. The DNA sequence coding for the 12-26 epitopewas modified by adding the coding sequence for the first 5 amino acidsof the framework region (FRI) of the VH coding sequence at the 3′ end ofthe synthetic DNA sequence coding for the 12-26 epitope. Thismodification allowed for proper folding and was selected to result inminimal disruption in the tertiary structure of the immunoglobulinmolecule. Regions of the Ig molecule that are likely to be sites whereinsertion of an epitope are not likely to disrupt the molecule can bedetermined by analyzing the amino acid sequence of the Ig molecule aswell as the tertiary structure. The N-terminal and CDR regions on the Igchain are preferable regions into which the epitopes can be inserted toresult in minimal disruption of the tertiary structure. Insertion at theN-terminal region allows for insertion of larger polypeptide≧10 kDa.

[0090] The modified 12-26 sequence including the sequence for the firstfive amino acids of the first framework region of the VH was obtainedvia polymerase chain reaction. A plasmid containing the 45 base pairnucleotide sequence coding for the 12-26 epitope was constructed bycloning the synthetic 45 base pair DNA sequence into the BamHI/ClaI siteof a plasmid pPX1647 containing the H-ld flagellin gene (provided by Dr.P. Brey, Praxis-Lederle Corp.), a derivative plasmid of pUC119. Themodified 12-26 sequence was amplified using PCR techniques and twoprimers.

[0091] The primers were designated OS-1 and OS-2. The primer OS-1contains the coding sequence for the PstI site and the coding sequencefor the first 5 amino acids of the 12-26 sequence. The sequence for OS-1(SEQ ID NO:3) is: 5′ TGATCTACTG CAGCTGGAGG ACGCGCGGCG G 3′.

[0092] The primer OS-2 was complementary to the coding sequence for thePstI site and to the coding sequence for the first 5 amino acids of thefirst framework region of VH and the last 6 amino acids of the 12-26sequence. The sequence for OS-2 (SEQ ID NO:4) is: 5′ CGACCTCCTGCAGTTGGACC TGCTTCTTCT TCTCGTATAT 3′.

[0093] The 82 bp product of the PCR method, i.e., the modified 12-26sequence, was isolated by high sieve agarose using standard methods.

[0094] The 82 base pair PCR fragment was digested with PstI to produce a65 bp fragment coding for the modified 12-26 epitope including the first5 amino acids of FRI. The 65 bp fragment was subcloned into the plasmidpBS at a pST1 site. The subcloning was done by digesting the modified12-26 sequence with PstI. The selected plasmids containing the PstIfragment of modified 12-26 were sequenced to confirm the presence ofthat fragment in proper orientation. Plasmids containing the modified12-26 sequence are referred to as pBS/12-26 and were sequenced toconfirm structure.

[0095] The modified 12-26 fragment from pBS/12-26 was subcloned intopBS/VH. The subcloning was performed by initially doing a partial PstIdigest of the pBS/VH to cut the VH region at a PstI site, which islocated at the coding sequence for the first framework amino acids 4 and5 of VH. The pBS/12-26 was fully digested with PstI. After ligation,plasmids containing the modified 12-26 sequence inserted after thecoding sequence for the first 5 amino acids of the first frameworkregion of the VH were selected by filter hybridization of bacterialcolonies using a p³² labeled 12-26 oligonucleotide as probe. Theresulting VH fusion sequence is as follows: L-FRI-12-26-FRI (L=leadersequence; FRI=the first 5 amino acids of the first framework region ofVH). Double stranded sequencing was done to confirm proper siteinsertion as well as orientation. These plasmids are designatedpBS/VH/12-26.

[0096] The presence of the VH/12-26 recombinant sequence in the plasmidwas verified by DNA sequencing methodologies. The VH DNA sequencesurrounding and including the modified 12-26 insert (SEQ ID NO:5) is asfollows: CAG GTC CAA CTG CAG CTG GAG GAC GCG CGG CGG CTG                    L   E   D   A   R   R   L AAG GCG ATA TAC GAG AAGAAG AAG CAG GTC CAA CTG K   A   I   Y   E   K   K   K CAG

[0097] The modified 12-26/VH recombinant from pBS/VH/12-26 was subclonedinto a plasmid pSV2-neo at the BamH1/ECORI sites. The pSV2-neo plasmidis derived from pSNR (Dr. Al Bothwell, Yale University New Haven, Conn.)and contains the V_(H) (NP-binding) inserted in an IgG_(I) heavy chain.The 8.5 kbp EcoRI fragment from pSNR-1 and which contains the constantregions 1-3 (CH 1-3) of α₁ chain was also subcloned into the pSV2-neo.Deletion of a 8.5 kbp region between the EcoRI sites of plasmid pSNR-1,which includes the CH1-3 coding sequence, was carried out using standardtechniques as disclosed in Current Protocols in Molecular Biology, Vol.1: Supplement 3.1.3, John Wiley & Sons (1989). The complete plasmidcontains the sequence coding for the variable heavy chain with the 65base pair sequence coding for the 12-26 epitope inserted at theN-terminus first framework region of the variable heavy chain and thesequence coding for the (CH1-3) constant regions 1-3. The orientation ofthe modified variable region sequence and the constant regions wereverified by Southern restriction analysis, as described in CurrentProtocols in Molecular Biology, cited supra. Successful recombinantswere selected by ampicillin and a large scale plasmid preparation wasgrown using standard methods.

EXAMPLE II Expression of Fusion Immunoglobulin p12-26 RecombinantConstructs

[0098] The recombinant plasmids containing the coding sequence for boththe VH/12-26 fusion and the CH1-3 of IgG1 were introduced into hostcells and expression of the fusion protein was detected. Transformationand detection of expression was carried out using standard methods asdescribed in Current Protocols in Molecular Biology, cited supra.

[0099] The 12-26 IgG1 DNA construct (Q3) as well as the control pSNRconstruct (P6) were electroporated into J558L myeloma cells whichsynthesize only λ light chains. Stable integrants were selected forgrowth in the presence of the antibiotic G418. Transfectomas expressingthe 12-26 IgG1 fusion protein were identified by analyzing cell culturesupernatants by Western blot and ELISA.

[0100] Transfectomas were grown to high density in serum-free media(RPMI-1640 with 5% FCS) in roller bottles and in bulk culture.Purification from serum-free transfectoma supernatants was accomplishedsuccessfully via binding with protein-A sepharose at pH 8, with elutionat pH 4, as well as with anti-mouse IgG affinity columns.

[0101] Purified supernatants from selected clones have been analyzed forexpression of 12-26 epitopes by Western blotting and ELISA by standardmethods. (See FIGS. 2 and 3.) For Western blotting, samples wereelectrophoresed on 10% SDS-PAGE. Gels were transferred to nitrocelluloseand probed with anti-mouse IgG (left lanes) or anti-12-26 monoclonalantibody B3.11 (right lanes) plus alkaline phosphatase-conjugatedantibodies as secondary reagents. The results are shown in FIG. 2. Onlythose cell culture supernatants from transfectomas containing the 12-26IgG1 construct (Q3) reacted with antibodies specific for mouse IgG (leftlanes) and antibodies specific for the 12-26 epitope the 12-26 epitope(right lanes).

[0102] For ELISA competitive inhibition assays, pre-titrated monoclonalantibody B3.11 was mixed with increasing amounts of 12-26 peptide, orthe 12-26 peptide chemically coupled to rabbit gamma globulin(RGG/12-26), or 12-26 IgG1 (Q3). The ability of the mixtures to bind toimmobilized 12-26 peptide was determined by standard methods. Theresults, shown in FIG. 3, indicate that the 12-26 IgG fusion protein wasable to effectively inhibit the binding of the monoclonal antibody tothe 12-26 epitope compared with the 12-26 peptide in solution.

[0103] The competitive inhibition ELISA studies show that these fusionimmunoglobulins can effectively compete with free synthetic peptide or12-26 chemically-conjugated to rabbit IgG for binding to monoclonalantibody anti-12-26 B3.11. In addition, the 12-26-IgG is immunogenic forthe 12-26 epitope when emulsified in CFA (data not shown). This suggeststhat the inserted peptide can be processed and presented in aphysiologically relevant manner even in the context of a self-IgGmolecule. Experiments also indicate that the 12-26 fusionimmunoglobulins can stimulate IL-2 production (measured by CTLL assay)in an H-2^(d) restricted 12-26 specific T-cell hybridoma (9C127) (datanot shown).

EXAMPLE III Tolerance Induction in Mice with the 12-26 IgG1 FusionProtein

[0104] A high dose pretreatment of animals with the 12-26 peptideinjected intravenously or intraperitoneally in saline or emulsified inincomplete Freund's adjuvant (IFA) can induce T-helper cell toleranceupon subsequent immunization with peptide in complete Freund's adjuvant(CFA). Scherer et al., Symp. on Quant. Biol., Cold Spring Harbor, N.Y.,54:497 (1989) Tolerance induction to the 12-26 epitope has beenconfirmed in T-cell proliferation assays. However, animals treated withpeptide are not tolerant at the B-cell level. That is, when challengedwith 12-26-flagellin (providing “carrier epitopes”), the response wasnot diminished (see below). This indicates the reductions with peptidechallenge were due to T- but not B-cell tolerance.

[0105] To determine whether the 12-26 IgG1 fusion protein can induceB-cell tolerance, the following experiment was conducted. Mouse spleencells were cultured in vitro in RPMI-1640+5% FCS for 18 hours. The mousespleen cells were then incubated with increasing concentrations ofeither free 12-26 peptide, a chemical conjugate of rabbit gamma globulinwith 12-26 (RGG-122-26) or with 12-26-IgG1 (Q3). At 18 hours, thesespleen cells were washed and then challenged with eitherlipopolysaccharide (a mitogenic stimulus, not shown) or the A29 fusionprotein of Salmonella flagellin that contains the 12-26 peptide. TheSalmonella flagellin fusion protein containing the 12-26 epitope hasbeen shown previously to be immunogenic both in vivo and in vitro (datanot shown). As a control for induction of tolerance, spleen cells weretreated with a rabbit anti-immunoglobulin previously shown to induceunresponsiveness in vitro. G. Warner et al., J. Immunol., 146:2185(1991). The effect of anti-Ig is shown as an open circle on the rightend of each graph. The responsiveness of the cells was measured byELISA. The results are shown as FIG. 4 (A29 fusion protein with 12-26peptide challenge).

[0106] The results indicate that when spleen cells are challenged withthe A29 fusion protein, the 12-26 IgG1 fusion protein (Q3.13), or thechemical conjugate (RGG-12-26) were both tolerogenic at microgramlevels. In contrast, the free peptide does not inhibit B-cellresponsiveness at any dose. Thus, these results indicate that the 12-26IgG fusion proteins can induce tolerance in B-cells in vitro. Similarresults were obtained in vivo as follows.

[0107] The 12-26-IgG fusion proteins were tested for induction oftolerance in vivo. CAF₁ mice were injected with 1 mg of the 12-26-IgGfusion protein, 12-26-IgG or free peptide in saline. Control micereceived PBS in saline. Spleen cells from these mice were challenged 10days later with the 12-26-flagellin fusion protein in vitro.Responsiveness to the 12-26 was measured by ELISA assays at 4 days afterchallenge as described for FIG. 4. The results are shown in FIG. 5.

[0108] The results indicate the 12-26 IgG fusion proteins as well as thechemical conjugate (RGG-12-26) can induce tolerance in vivo and invitro. See FIGS. 4 and 5.

EXAMPLE IV Construction of Retroviral Vector Containing a DNA SequenceCoding for the 12-26 IgG1 Fusion Protein

[0109] Several retroviral constructs have been prepared that are basedon the murine Moloney leukemia retroviral vector MBAE , as described byKang et al., Proc. Natl. Acad. Sci., 87:9803 (1990)

[0110] The retroviral vector MBAE can be obtained from Dr. Hozumi orprepared as described by Kang et al., cited supra. Briefly, theretroviral vector containing the Moloney murine leukemia long terminalrepeats and the neo gene coding for G418 resistance was modified byinsertion of the β-actin promoter and enhancer sequences. The β-actinpromoter and enhancer sequences were cloned downstream from the neogene. Heterologous genes can then be inserted downstream from theβ-actin promoter by subcloning with HindIII and SalI.

[0111] DNA sequences subcloned into MBAE were derived from PCR-amplifiedreverse transcribed RNA from transfectoma Q3 which contains the12-26-IgG H chain. The Q3 transfectoma was prepared as described inExample II. The RNA from the Q3 transfectoma was harvested and incubatedwith reverse transcriptase in a standard PCR reaction as described inCurrent Protocols in Molecular Biology, cited supra. to form cDNAmolecules. The cDNA molecules were amplified using the followingprimers: V₈ 5′ primer (SEQ ID NO:6): 5′ TGG ACT AAG TCG ACA CCA TGG GATGCA GC pep 3′ primer (SEQ ID NO:7): 5′ GGC AAC AGA AGC TTT CAC TTC TTCTTC TCG TAT 3′

[0112] One such cDNA includes a DNA sequence coding for the leadersequence and the 12-26 epitope from the variable heavy chain genefollowed by a stop codon. The stop codon was designed into the PCRprimer at the end of the DNA sequence coding for the last amino acid of12-26 (in primer) to construct a peptide minigene.

[0113] A DNA sequence coding for the leader sequence and the sequencecoding for the 12-26 epitope followed by a stop codon was subcloned intopBluescript and sequenced and then subcloned into the MBAE vector.Subcloning was performed using SalI and HindIII to insert the peptideminigene downstream from the β-actin promoter and enhancer sequences, asshown in FIG. 7.

[0114] The recombinant MBAE vectors were transfected by lipofection intothe ψ-2 cell line available from Dr. N. Hozumi (Toronto, Canada). Thetransfected cells lines were grown in RPMI 5% FCS in the presence of 0.8mg/ml crude G418 . G418 resistent clones were isolated by limitingdilution and viral titer was determined on NIH 3T3 cells in the presenceof 0.8 mg/ml G418 (crude weight). For the peptide minigene construct,one transfected ψ-2 clone (MBAE pEP19) with a titer of 10⁵-10⁶ CFU/mlwas chosen for subsequent gene transfer experiments. Presence of helpervirus was assayed using standard methods (“horizontal spread ofinfection” method), as described by Current Protocols in MolecularBiology, cited supra. and was not detected. Virus producing lines werethawed out fresh for each individual experiment.

[0115] An A20.2J B-cell lymphoma cell, available from ATCC, infectedwith the viral vector expressed and secreted the peptide as detected byWestern blot. See FIG. 6A. After infection of A20.2J B-cell lymphomacells, the cells were grown in G418 and 200 μl of supernatants wereanalyzed by Western blotting. Supernatants from four ψ-2/A20.2J clonesinfected with retroviral 12-26 minigene were slot blotted and reactedwith monoclonal antibody B3.11 specific for the 12-26 epitope. As seenin FIG. 6A, the peptide was expressed in the infected lymphoma cells.

[0116] The A20.2J infected cells not only produce the peptide but alsopresent it to a 12-26 reactive T-cell hybridoma. Briefly, titratedvolumes of supernatants from infected A20.2J cells were incubated with a12-26 reactive T-cell clones (T32) for 24-48 hours. The 12-26 reactiveT-cell clones was obtained by Dr. Tom Briner and Dr. M. Gefter(Massachusetts Institute of Technology, Cambridge, Mass.).Responsiveness of the T-cell clone was measured by ³H-thymidineincorporation and standard IL-2 assay. The results are shown in FIG. 6B.The results indicate that A20 cells process this peptide so it can bepresented to a 12-26 reactive T-cell clone. IL-2 production by theseclones was also measured and the results show the 12-26 peptide isproduced and secreted by the infected cells.

EXAMPLE V Preparation of Mice Carrying Transfected Bone Marrow Cells

[0117] Mice carrying bone marrow cells transfected with the viral vectorMBAE 12-26 coding for the 12-26 epitope (FIG. 7) were prepared. Bonemarrow progenitors from Balb/c mice were infected with the MBAE 12-26vector as described by Chambers et al., Proc. Natl. Acad. Sci., 89:1026(1992). Marrow donor Balb/c mice were pretreated intravenously with 150mg/kg 5-fluorouracil for 3-4 days before marrow harvest. Fractionatedmarrow cells were kept on ice and then washed in complete RPM1 with 15%FC5 and 10 units/ml IL-3. The bone marrow cells were then coculturedwith about an 80% confluent layer of irradiated (2000 rads) ψ-2packaging lines. Co-culture with adherent ψ-2 virus producing line wasdone at 37° C. for 48 hours as follows:

[0118] 5×10⁶ marrow cells per 6 wells in 10 ml medium containing:

[0119] 15% FCS

[0120] 6 μg/ml polybrene

[0121] 100 units/ml IL-6

[0122] 200 units/ml IL-3

[0123] Nonadherent bone marrow cells were harvested after 48 hours,washed and resuspended in HEPES buffered Eaglis medium. Syngeneicrecipient Balb/c mice were lethally irradiated with 900 rads and 4×10⁶cells in a volume of 400 μl were injected into the irradiated miceintravenously. Recipient mice were started on acidified water 1-2 weeksbefore transplantation to prevent gram negative infections andmaintained in autoclaved microisolater cages with autoclaved food,bedding and acidified water supplemented with antibiotics.

[0124] After two weeks, the lymphoid cells from the recipient mice wereharvested from tail bleeds and examined for the presence of the 12-26sequence by RT-PCR. Fragments of about 100 base pairs were detected inboth infected lymphoid cells and the ψ-2 MBAE 12-26 containing cellline. See FIG. 8.

[0125] Briefly, RNA from peripheral blood cells taken from the animalsat 2 weeks or from infected ψ-2 packaging lines was reverse transcribed.DNA sequences coding for the 12-26 epitope were amplified using theV_(H) (SEQ ID NO:6) and pep (SEQ ID NO:7) primers. Amplified productswere separated by agarose gel electrophoresis and products containing aDNA sequence coding for the 12-26 epitope were detected by Southernblot. The probe used to detect 12-26 coding sequences is as follows (SEQID NO:8): 5′ - TGATCTACTG CAGCTGGAGG ACGCGCGGCG G - 3′

[0126] Hybridization was conducted under standard conditions asdescribed in Current Protocols, cited supra. A fragment detected inperipheral blood cells by hybridization to 12-26 probe indicatedexpression of the 12-26 epitope was occurring in the cells 2 weeks afteradministration.

What is claimed is:
 1. An expression vector for persistently maintainingexpression of an tolerogenic epitope in an animal comprising: (a) a DNAsequence coding for a fusion immunoglobulin operably linked totranscriptional and translational control regions functional in ahemopoietic cell or lymphoid cell, wherein the fusion immunoglobulin hasat least one heterologous tolerogenic epitope at the N-terminus variableregion; and wherein said DNA sequence is operably linked to (b) a vectorthat can provide for stable maintenance of the DNA sequence in thehemopoietic cell or lymphoid cell.
 2. An expression vector according toclaim 1, wherein the vector is a retroviral vector.
 3. An expressionvector according to claim 1, wherein the DNA sequence codes for a fusionIgG having a heterologous tolerogenic epitope inserted adjacent to thefirst framework region of the N-terminus variable region of the heavychain.
 4. An expression vector according to claim 3, wherein the DNAsequence encodes a fusion IgG including an epitope having the amino acidsequence of amino acids 12-26 of the λ CI repressor protein.
 5. Anexpression vector according to claim 1, wherein the transcriptional andtranslational control regions provide for constitutive expression of theDNA sequence in the lymphoid cells.
 6. A method for tolerizing an animalto an epitope comprising: (a) providing a vector that can be stablymaintained in a hemopoietic or lymphoid cell, wherein the vectorcomprises a DNA sequence that codes for a fusion immunoglobulin operablylinked to transcriptional and translational control regions functionalin the hemopoietic or lymphoid cell, wherein the fusion immunoglobulinhas at least one heterologous tolerogenic epitope at the N-terminusvariable region; (b) stably transforming a population of the hemopoieticor lymphoid cells from the animal with the vector to form a transformedpopulation of hemopoietic or lymphoid cells expressing the fusionimmunoglobulin; and (c) introducing the transformed population of cellsinto an animal.
 7. A method according to claim 6, wherein the fusionimmunoglobulin has a tolerogenic epitope having the amino acid sequenceof amino acids 12-26 of the λ CI repressor protein, wherein thetolerogenic epitope is inserted at the first framework region of theN-terminus of the variable heavy chain.
 8. A method according to claim7, wherein the vector is a retroviral vector.
 9. A method according toclaim 6, further comprising irradiating the animal sufficiently todestroy endogenous hemopoietic cells before introducing the transformedhemopoietic cells into the animal.
 10. An expression cassette forexpression of a DNA sequence in a hemopoietic or lymphoid cellcomprising: (a) a DNA sequence coding for a fusion immunoglobulinwherein the fusion immunoglobulin has at least one heterologoustolerogenic epitope inserted adjacent to the first framework region atthe N-terminus of the variable region of the immunoglobulin, operablylinked to transcriptional and translational control regions functionalin the hemopoietic or lymphoid cells.
 11. An expression cassetteaccording to claim 10, wherein the epitope has the amino acid sequenceof amino acids 12-26 of the λ CI repressor protein.
 12. An expressioncassette according to claim 11, wherein the fusion immunoglobulin is anIgG.
 13. A plasmid having the characteristics of ATCC No. .
 14. Apharmaceutical composition comprising: (a) a tolerogenic amount of afusion immunoglobulin wherein the fusion immunoglobulin has at least oneheterologous tolerogenic epitope adjacent to the first framework regionof the N-terminus variable chain; and (b) a pharmaceutically acceptableexcipient.
 15. A pharmaceutical composition according to claim 14,wherein the pharmaceutical immunoglobulin is an isologous IgG.
 16. Apharmaceutical composition according to claim 15, wherein the fusionimmunoglobulin has an heterologous tolerogenic epitope with an aminoacid sequence of amino acids 12-26 of the λ CI repressor protein.
 17. Apharmaceutical composition, wherein the excipient is selected from thegroup consisting of phosphate buffered saline, physiological saline andwater.
 18. A pharmaceutical composition, wherein the tolerogenic amountof the fusion immunoglobulin is about 4 to 40 mg/kg of body weight ofthe animal.
 19. A transformed hemopoietic or lymphoid cell comprising anexpression cassette stably maintained in the hemopoietic or lymphoidcell, wherein the expression cassette comprises a DNA sequence codingfor a fusion immunoglobulin, wherein the fusion immunoglobulin has atleast one heterologous tolerogenic epitope inserted adjacent to thefirst framework region at the N-terminal variable region, wherein saidDNA sequence is operably linked to transcriptional and translationalcontrol regions functional in the hemopoietic or lymphoid cell.
 20. Atransformed cell according to claim 19, wherein the cell is a bonemarrow cell.
 21. A method for identifying tolerogenic epitopescomprising: (a) providing a vector that can be stably maintained in ahemopoietic or lymphoid cell, wherein the vector comprises a DNAsequence that codes for a fusion immunoglobulin operably linked totranscriptional and translational control regions functional in thehemopoietic or lymphoid cell, wherein the fusion immunoglobulin has atleast one heterologous epitope at the N-terminus variable region; (b)stably transforming a population of hemopoietic or lymphoid cells froman animal with the vector to form a population of transformed cells; (c)introducing the transformed cells into an animal; and (d) identifyingwhether the heterologous epitope is a novel tolerogen by determiningwhether the animals are tolerant to the heterologous epitope.
 22. Amethod of identifying tolerogenic epitopes comprising; (a) providing avector that can be stably maintained in a host cell, wherein the vectorcomprises a DNA sequence that codes for a fusion immunoglobulin operablylinked to transcriptional and translational control regions functionalin the host cell, wherein the fusion immunoglobulin has at least oneheterologous epitope at the N-terminus variable region of theimmunoglobulin; (b) stably transforming a population of host cells withthe vector to form a population of transformed cells, producing thefusion immunoglobulins; and (c) identifying whether the heterologousepitope on the fusion immunoglobulin is a tolerogenic epitope bydetermining whether the epitope is associated with an autoimmune orallergic immune response.
 23. A method according to claim 22, whereinthe host cell is E. coli.
 24. A method according to claim 22, whereinthe vector is a phagemid vector.
 25. A method according to claim 22,wherein the host cell is a J558L cell.
 26. A method according to claim22, wherein the step of identifying whether the heterologous epitope onthe fusion immunoglobulin is a tolerogen comprises: (a) determiningwhether the fusion immunoglobulin immunoreacts with immune serum from anautoimmune or allergic animal.
 27. A method according to claim 22,wherein the step of identifying whether the heterologous epitope on thefusion immunoglobulin is a tolerogen comprises: (a) determining whetherthe fusion immunoglobulin stimulates proliferation of lymphocytes froman autoimmune or allergic animal.
 28. A method according to claim 22further comprising: (a) confirming that the heterologous epitope is atolerogenic epitope by determining whether the fusion immunoglobulininduces tolerance to the epitope in an animal.
 29. A method oftolerizing an animal to an epitope comprising: administering a fusionimmunoglobulin having a heterologous tolerogenic epitope to an animalsufficiently to induce tolerance to the heterologous tolerogenicepitope, wherein the fusion immunoglobulin has the heterologoustolerogenic epitope at the first N-terminus framework region of theimmunoglobulin.
 30. A method of inducing and maintaining tolerance to anepitope in an animal comprising: (a) administering a pharmaceuticalcomposition according to claim 14 sufficiently to induce tolerance to anepitope; and (b) administering transformed hemopoietic or lymphoid cellsto the animal sufficiently to maintain tolerance to the epitope, whereinthe transformed cell comprises a vector stably maintained in thetransformed cell, wherein the vector comprises a DNA sequence coding fora fusion immunoglobulin operably linked to transcriptional andtranslational control regions functional in the cell, wherein the fusionimmunoglobulin has at least one heterologous tolerogenic epitope at theN-terminus variable region of the immunoglobulin.